1. Field of the Invention.
Significant morbidity and mortality are associated with infectious diseases. More rapid and accurate diagnostic methods are required for better monitoring and treatment of disease. Molecular methods using DNA probes, nucleic acid hybridizations and in vitro amplification techniques are promising methods offering advantages to conventional methods used for patient diagnoses.
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the .sup.32 P labeling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient's body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
One method for detecting specific nucleic acid sequences generally involves immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labeled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
When very low concentrations must be detected, the above method is slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable.
Recently, a method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other, leading to the accumulation of a discrete fragment whose length is defined by the distance between the 5' ends of the oligonucleotide primers.
Other methods for amplifying nucleic acids are single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q-beta-replicase method. Regardless of the amplification used, the amplified product must be detected.
For any of the above methods for amplifying nucleic acid there is a risk of contaminating the amplification mixture with previously amplified material and thereby amplifying material that was not present in the original sample, namely, a contaminant. The quantities of amplification product can be very large thereby aggravating the potential contamination. Once aerosols of amplified nucleic acid are produced in a laboratory, droplets containing this material can invade subsequent amplification mixtures or equipment. Attempted amplification of a nucleic acid may then produce amplified copies of this contaminating material even when the target nucleic acid, or sequence thereof, was not present in the sample being amplified. Such contamination can also occur if the same container is employed for multiple amplifications even though the container is cleaned. As few as one molecule will sometimes be sufficient to contaminate other containers that are to be used in further amplifications. This possibility for contamination can result in a false test since such a single molecule can be amplified and detected. The result of the test will not accurately reflect the presence or absence of the particular nucleic acid in the patient sample being tested.
After amplification of a particular nucleic acid, a separate step is carried out prior to detecting amplified material. One method for detecting nucleic acids is to employ nucleic acid probes. One method utilizing such probes is described in U.S. Pat. No. 4,868,104. A nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support. Detection of signal depends upon the nature of the label or reporter group. If the label or reporter group is an enzyme, additional members of the signal producing system include enzyme substrates and so forth.
It is desirable to have a sensitive, simple method for amplifying and detecting nucleic acids preferably, in a homogeneous format. The method should minimize the number and complexity of steps and reagents. The need for sterilization and other steps needed to prevent contamination of assay mixtures should be avoided.
2. Description of the Related Art.
Rapid, non-separation electrochemiluminescent DNA hybridization assays for PCR products using 3'-labeled oligonucleotide probes is described by Gudibande, et al., (1992) Molecular and Cellular Probes, 6: 495-503. A related disclosure is found in international patent application WO 9508644 A1 (950330).
Marmaro, et al.,(Meeting of the American Association of Clinical Chemists, San Diego, Calif., November 1994, Poster No. 54) discusses the design and use of fluorogenic probes in TaqMan, a homogeneous PCR assay.
A PCR-based assay that utilizes the inherent 5' nuclease of rTth DNA polymerase for the quantitative detection of HCV RNA is disclosed by Tsang, et al., (94th General Meeting of the American Society for Microbiology, Las Vegas Nev. 5/94, Poster No. C376).
Kemp, et al., (1990) Gene, 94:223-228, disclose simplified colorimetric analysis of polymerase chain reactions and detection of HIV sequences in AIDS patients.
German patent application DE 4234086-A1 (92.02.05) (Henco, et al.) discusses the determination of nucleic acid sequences amplified in vitro in enclosed reaction zone where probe(s) capable of interacting with target sequence is present during or after amplification and spectroscopically measurable parameters of probe undergo change thereby generating signal.
U.S. Pat. No. 5,232,829 (Longiaru, et al.) discloses detection of chlamydia trachomatis by polymerase chain reaction using biotin labeled DNA primers and capture probes. A similar disclosure is made by Loeffelholz, et al. (1992) Journal of Clinical Microbiology, 30(11):2847-2851.
Padlock probes: circularizing oligonucleotides for localized DNA detection are described by Nilsson, et al. (1994) Science, 265:2085-2088.
A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182. Sequence polymerization by polymerase chain reaction is described by Saiki, et al., (1986) Science, 230: 1350-1354. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al., Science (1988) 239:487.
U.S. patent application Ser. Nos. 07/299,282 and 07/399,795, filed Jan. 19, 1989, and Aug. 29, 1989, respectively, describe nucleic acid amplification using a single polynucleotide primer (ASPP). U.S. patent application Ser. No. 07/555,323 filed Jul. 19, 1990, discloses methods for producing a polynucleotide for use in single primer amplification. U.S. patent application Ser. No. 07/555,968 describes a method for producing a molecule containing an intramolecular base-pair structure. A method for producing a polynucleotide for use in single primer amplification is described in U.S. patent application Ser. No. 07/776,538 filed Oct. 11, 1991. A method for introducing defined sequences at the 3'-end of a polynucleotide is described in U.S. patent application Ser. No. 08/140,369, filed Oct. 20, 1993. The disclosures of these six applications are incorporated herein by reference including the references listed therein in the sections entitled "Description of the Related Art."